The invention relates to a method of determining blood groups with blood-group specific immunoglobulin-M antibodies and to a support and a kit for carrying out the method.
The agglutination reaction has, due to its simplicity and wide range of application, become one of the most common methods of determining blood groups in recent decades. One of its essential drawbacks however is that it lacks an objective terminal point that can easily be registered either automatically or visually because the reactions that occur within the dilution series vary in intensity to the extent that weak reactions for example can be incorrectly considered negative.
The ELISA (enzyme-linked immunosorbent assay) has recently been employed to determine blood groups. Although this method is admittedly extremely sensitive, it also has drawbacks in the number of steps it requires and in problems relating to the absorbability of antibody and enzyme by erythrocytes and to interactions with intercellular hemoglobin in solution.
Solid-phase methods have recently been introduced as alternatives to the agglutination reaction and to the ELISA test for determining blood groups in the ABO and Rh systems, for antibody screening and for cross-matching (1: H. L. Moore et al., Transfusion 22, 540 [1982]; 2: H. H. Moore, Transfusion 24, 218-221 [1984]; 3: L. T. Sinor et al., Transfusion 25, 21-23 [1985]; 4: F. V. Plapp et al., A.J.C.P. 82, 719-721 [1984]; 5: M. L. Beck et al., Med. Lab. Sci. 41, 374-381 [1984]; 6: J. M. Rachel et al.,
25, 24-26 [1985]; and 7: F. V. Plapp et al., The Lancet [1986], 1465-1466). Common to these methods is that positive reaction patterns are represented by a layer of specifically attached erythrocytes on specially prepared solid phases, whereas negative reactions do not lead to the formation of a solid-phase layer. The varying reaction patterns can be read visually or spectrophotometrically, leading to an objective "yes" or "no."
Solid-phase methods of ABO and Rh determination have previously been carried out by exposing a suitable support, such as microtitration plates made of polyvinyl chloride (2) or polystyrene (3) or membranes of nylon or nitrocellulose (7) to buffer solutions of monoclonal or affinity-purified antibodies for long periods, at least 2 hours (2) for example, for coating, leading to the formation of immobilized, meaning irreversibly attached monomolecular solid-phase layers on the plastic surfaces due to passive adsorption. Excess antibodies are rinsed away and the supports, prepared for use in this way, are available for blood-group serology study. The use of anti-D of the IgG type for Rh determination required the creation of a double solid-phase layer consisting of affinity-purified anti-human-IgG (3 & 5) and anti-D to overcome the distance between the plastic surface and the offered erythrocytes.
Although the solid-phase methods known up to now have considerable advantages over the agglutination and ELISA methods, the preliminary coating of the surface of the support has always been complicated.